Synaptic transmission occurs through the release of neurotransmitter from vesicles and the subsequent activation of a postsynaptic voltage change. It underlies every aspect of brain function and is relevant to neurological diseases. The key step in synaptic transmission, vesicle fusion, is dependent on Ca2+ entry through presynaptic voltage-gated Ca2+ channels (VGCCs) that are activated by an action potential. However, despite many years of study, the role of VGCCs in regulating spontaneous vesicle fusion in the absence of an action potential is not clear. VGCCs are tightly coupled to the release machinery for spontaneous release of the inhibitory neurotransmitter GABA in cortical neurons, and multiple VGCCs cooperate in triggering spontaneous release at these synapses. Additionally, activation of the Ca2+-sensing receptor (CaSR) by Ca2+ facilitates spontaneous GABA release. The goal of this proposal is to improve understanding of the role of Ca2+ in regulating neurotransmitter release, specifically how it is different at different types of synapse and for different forms of release. The overall hypothesis is that spontaneous and action potential-dependent release of GABA are regulated by Ca2+ via distinct mechanisms. This hypothesis will be tested in neocortical neuronal cultures using whole-cell voltage-clamp recordings of inhibitory postsynaptic currents to detect release of GABA and Ca2+ imaging to monitor changes in intraterminal Ca2+. Aim 1 is to determine how VGCCs regulate GABA release. The first experiment in this proposal will use Ca2+ chelators, EGTA and BAPTA, to test if the diffusion distance for calcium from VGCCs to the vesicle release machinery for activity-evoked release of GABA is different from that observed for spontaneous GABA release. The next experiment in this aim will use Ca2+ imaging to determine if changes in the extracellular Ca2+ concentration produce changes in the intraterminal Ca2+ concentration in GABAergic neurons. The final experiment in this aim will investigate the mechanism underlying VGCC cooperativity for spontaneous GABA release. This could occur through two possible mechanisms: (i) physiological coupling through linkage of multiple channels or (ii) stochastic synchronized gating. Both of these possibilities will be investigated by measuring VGCC cooperativity for spontaneous GABA release in cultured neurons from VGCC knock-out mice that are transfected with mutant VGCCs, lacking their C-terminal tails, and computer modeling and Monte Carlo simulation. Aim 2 will determine how the CaSR regulates spontaneous GABA release. These experiments will use pharmacological tools and intracellular Ca2+-imaging to determine the players in the CaSR-signaling pathway. Together, this proposal will provide an understanding of the role of Ca2+ in regulating synaptic transmission. Understanding the mechanism by which Ca2+ influences release of neurotransmitter will contribute to an improved understanding of synaptic function in general and when transmission is disrupted in disease states.